The generation of an immune response involves the sensitization of helper (CD4+) (TH) and cytotoxic (CD8+) (CTL) T cell subsets through their interaction with antigen presenting cells. Antigen presenting cells express major histocompatibility (MHC)-class I or class II molecules associated with antigenic fragments (i.e., specific amino acid sequences derived from an antigen which bind to MHC I and MHC II for presentation on the cell surface). The MHC in humans is also referred to as the HLA (human leukocyte antigen) complex. The sensitized CD4+ T cells produce lymphokines that participate in the activation of B cells as well as various T cell subsets. The sensitized CD8+ T cells increase in numbers in response to lymphokines and act to destroy cells that express the specific antigenic fragments associated with matching MHC-encoded class I molecules. In the course of a tumor or viral infection, cytotoxic T cells eradicate cells expressing tumor or virus associated antigens.
Dendritic cells (DCs) are thought to be the most potent antigen presenting cells of the immune system (reviewed in Steinman, R. M. 1991. The dendritic cells system and its role in immunogenicity. Ann. Rev. Immunol. 9:271; Banchereau, J. B. and R. M. Steinman. 1998. Dendritic cells and the control of immunity. Nature. 392:245). Given their broad spectrum of roles in initiating the immune response by internalizing and processing antigens, migrating to lymphoid organs, secreting cytokines, and expressing co-stimulatory molecules required for lymphocyte signaling, it is no surprise that dendritic cells are logical targets for clinical use (Banchereau, J. B. and R. M. Steinman. 1998. Dendritic cells and the control of immunity. Nature. 392:245). By targeting antigens into dendritic cells in vivo or exposing dendritic cells to antigen ex vivo, it may be possible to enhance the immunogenicity of vaccines by eliciting helper and cytotoxic T cells, antibodies, and IL-12 for prophylactic applications, or induce T cell mediated anti-tumor responses for cancer immunotherapy. Akbari, et al. have suggested that transfection and activation of dendritic cells are key events for immunity following DNA vaccination by scarification of the ear skin in mouse models (O. Akbari, N. P., S. Garcia, R. Tascon, D. Lowrie, and B. Stockinger. 1999. DNA vaccination: transfection and activation of dendritic cells as key events for immunity. J. Exp. Med. 189:169). Anti-tumor CTL activity and protection against lethal tumor challenge in mouse models have been demonstrated using cytokine-driven bone-marrow-derived dendritic cells (BMDCs) pulsed with tumor-associated peptides (J. I. Mayordomo, T. Z., W. J. Storkus. 1995. Bone marrow-derived dendritic cells pulsed with synthetic tumour peptides elicit protective and therapeutic antitumour immunity. Nature Med. 1:1297), and whole tumor lysates (R. C. Fields, K. S., and J. J. Mule'. 1998. Murine dendritic cells pulsed with whole tumor lysates mediate potent antitumor immune responses in vitro and in vivo. Proc. Natl. Acad. Sci. USA 95:9482) transferred by the subcutaneous route.
In vitro generation of dendritic cells has been optimized sufficiently so that genetic immunotherapy based on passive transfer of dendritic cells has become an attractive target for development (N. Romani, S. G., D. Brang. 1994. Proliferating dendritic cell progenitors in human blood. J. Exp. Med. 180:83). However, in vitro transfection efficiency of dendritic cells by non-viral methods has been extremely poor (J. F. Arthur, L. H. B., M. D. Roth, L. A. Bui, S. M. Kiertscher, R. Lau, S. Dubinett, J. Glaspy, W. H. McBride, and J. S. Economou. 1997. A comparison of gene transfer methods in human dendritic cells. Cancer Gene Ther. 4:17) and has limited progress toward effective dendritic-cell-based immunotherapy. While progress has been made by the use of electroporation, the efficiency of transfection is extremely low and results in substantial loss of cell viability (V. F. I. Van Tendeloo, H.-W. S., F. Lardon, GLEE Vanham, G. Nijs, M. Lenjou, L. Hendriks, C. Van Broeckhoven, A. Moulijn, I. Rodrigus, P. Verdonk, D. R. Van Bockstaele, and Z. N. Berneman. 1988. Nonviral transfection of distinct types of human dendritic cells: high efficiency gene transfer by electroporation into hematopoietic progenitor- but not monocyte-derived dendritic cells. Gene Ther. 5:700). To date, no purely chemical method has been shown to be effective.
Particulate carriers have been used in order to achieve controlled, parenteral delivery of therapeutic compounds. Such carriers are designed to maintain the active agent in the delivery system for an extended period of time. Examples of particulate carriers include those derived from polymethyl methacrylate polymers, as well as microparticles derived from poly(lactides) (see, e.g., U.S. Pat. No. 3,773,919), poly(lactide-co-glycolides), known as PLG (see, e.g., U.S. Pat. No. 4,767,628) and polyethylene glycol, known as PEG (see, e.g., U.S. Pat. No. 5,648,095). Polymethyl methacrylate polymers are nondegradable while PLG particles biodegrade by random nonenzymatic hydrolysis of ester bonds to lactic and glycolic acids, which are excreted along normal metabolic pathways.
For example, U.S. Pat. No. 5,648,095 describes the use of microspheres with encapsulated pharmaceuticals as drug delivery systems for nasal, oral, pulmonary and oral delivery. Slow-release formulations containing various polypeptide growth factors have also been described. See, e.g., International Publication No. WO 94/12158, U.S. Pat. No. 5,134,122 and International Publication No. WO 96/37216.
Particulate carriers have also been used with adsorbed or entrapped antigens in attempts to elicit adequate immune responses. Such carriers present multiple copies of a selected antigen to the immune system and promote trapping and retention of antigens in local lymph nodes. The particles can be phagocytosed by macrophages and can enhance antigen presentation through cytokine release. For example, commonly owned, co-pending application Ser. No. 09/015,652, filed Jan. 29, 1998, describes the use of antigen-adsorbed and antigen-encapsulated microparticles to stimulate cell-mediated immunological responses, as well as methods of making the microparticles.
In commonly owned provisional Patent Application 60/036,316, for example, a method of forming microparticles is disclosed which comprises combining a polymer with an organic solvent, then adding an emulsion stabilizer, such as polyvinyl alcohol (PVA), then evaporating the organic solvent, thereby forming microparticles. The surface of the microparticles comprises the polymer and the stabilizer. Polynucleotides such as DNA, polypeptides, and antigens may then be adsorbed on those surfaces. See also PCT US99/17308.
Commonly owned Provisional Application No. 60/146,391 discloses a method of forming microparticles with adsorbent surfaces that are capable of adsorbing a variety of macromolecules including polynucleotides. In one embodiment, the microparticles are comprised of both a polymer and a detergent. The microparticles are derived from a polymer, such as a poly(α-hydroxy acid), preferably, a poly(D,L-lactide-co-glycolide), a polyhydroxy butyric acid, a polycaprolactone, a polyorthoester, a polyanhydride, a polycyanoacrylate, and the like, and are formed with detergents, such as cationic, anionic, or nonionic detergents, which detergents may be used in combination. Cationic detergents disclosed are cetrimide (CTAB), benzalkonium chloride, DDA (dimethyl dioctodecyl ammonium bromide), DOTAP, and the like. It is noted that these microparticles yield improved adsorption of viral antigens, and provide for superior immune responses, as compared to microparticles formed by a process using only PVA.
Dendritic cells can capture antigen at peripheral sites via macropinocytosis using membrane ruffling, or may also internalize antigen by receptor-mediated processes involving FcγIII, the mannose receptor, or the C-type lectin DEC-205 (reviewed in Lanzavecchia, A. 1996. Mechanisms of antigen uptake for presentation. Curr. Op. Immunol. 8:348). Thus, dendritic cells may be targeted by the capture of larger (>250 nm) particulate antigens by phagocytosis. Biodegradable polymer microspheres such as poly-lactide-co-glycolide (PLG) are readily internalized by phagocytic cells up to a diameter of 5 μm (Ikada, Y. T. et al. 1990. Phagocytosis of polymer microspheres by macrophages. Adv. Polymer. Sci. 94:107) and have been utilized as carriers for drug delivery systems.
Recently, Newman, et al. reported cytoplasmic delivery of Texas red labeled dextran encapsulated in PLGA microspheres following phagocytosis in mouse peritoneal macrophages (K. D. Newman, G. K., J. Miller, V. Chlumecky, J. Samuel. 1999. Cytoplasmic delivery of a fluorescent probe by poly(D,L lactic-co-glycolic acid) microspheres. In 1999 AAPS Annual Meeting Abstracts Online, vol. 1).
The application of synthetic biopolymers for nucleic acid delivery has proven advantageous by protecting DNA against nuclease degradation and increasing cellular uptake (C. Chavany, T. S.-B., T. Le Doan, F. Puisieux, P. Couvreur, and C. Helene. 1994. Adsorption of oligonucleotides onto polyisohexylcyanoacrylate nanoparticles protects them against nucleases and increases their cellular uptake. Pharm. Res. 11:1370).
Evidence for direct transfection of non professional antigen presenting cells mediated by PLG was recently reported by Ciftci and Su who found PLG microparticles containing a DNA:polycation complex provided controlled release of DNA and surfactant-enhanced uptake and gene expression in 293 and MCF-7 cells (K. Ciftci, J. S. 1999. DNA-PLGA microparticles: a promising delivery system for cancer gene therapy. In 1999 AAPS Annual Meeting Abstracts Online, vol. 1).
While polyalkylcyanoacrylate nanoparticles have been used to bind CTAB-oligonucleotide complexes to deliver antisense oligonucleotides to macrophage cell lines in vitro (C. Chavany, T. S.-B., T. Le Doan, F. Puisieux, P. Couvreur, and C. Helene. 1994. Adsorption of oligonucleotides onto polyisohexylcyanoacrylate nanoparticles protects them against nucleases and increases their cellular uptake. Pharm. Res. 11:1370; E. Fattal, C. V., I. Aynie, Y. Nakada, G. Lambert, C. Malvy, and P. Couvreur. 1998. Biodegradable polyalkylcyanoacrylate nanoparticles for the delivery of oligonucleotides. J. Controlled Release 53:137), these vehicles have not been shown to transfect dendritic cells with plasmids carrying recombinant genes.
Hence, there is a need in the art for an effective non-viral technique for the transfection of dendritic cells. While microparticle technology has been heretofore used for introduction of polynucleotides into cells, applicants are aware of no such technology having been used for the transfection of dendritic cells, which are notoriously resistant to transfection.